So far, genes encoding a hormone or growth factor have been isolated and utilized to produce many recombinant proteins that are commercialized as medicines. Most of them are secretory proteins. Therefore, isolation of a gene encoding a novel secretory protein is an extremely important step in developing a novel medicine. Accordingly, methods for isolating a gene encoding a secretory protein have been developed. For example, Honjo et al. developed a method (unexamined published Japanese patent application No. Hei 6-315380) by utilizing the feature that secretory proteins have a signal sequence that allows intracellularly expressed proteins to translocate to the cell surface. In this method, the signal sequence of the α chain of human IL-2 receptor, a secretory protein, is replaced with a short cDNA fragment corresponding to the 5′-end sequence of mRNA from a target cell or tissue to construct a library, which is then introduced into cells. Among the clones, IL-2 receptor is expressed on the cell surface of clones with a signal sequence, but not those without a signal sequence. The presence of the signal sequence can thus be detected by the anti-IL-2 receptor antibody.
Genetics Institute, Inc., (Cambridge, Mass.) developed a more sophisticated system utilizing a yeast metabolic enzyme (U.S. Pat. No. 5,536,637). Invertase, a yeast metabolic enzyme, is a secretory enzyme that cleaves sucrose in the culture medium to glucose and fructose to transfer energy. A mutant strain that does not secrete this enzyme can not grow in a medium containing sucrose as the sole carbon source without glucose. In this method, which utilizes the phenomenon, invertase gene is ligated with cDNA to construct a library, which is then introduced into a mutant yeast strain lacking the invertase gene. Clones containing the signal peptide are isolated by selecting clones capable of growing in a medium containing only sucrose as a carbon source.
However, the method of Honjo et al. is disadvantageous in that laborious steps are required in selecting positive clones due to the use of an antibody. Furthermore, the detection sensitivity is very low. The method of Genetics Institute, Inc., also has a problem in that a clone with poor secretion efficiency in yeast cannot be isolated. In addition, these methods detect only short DNA because of the potential loss of antigenicity or enzymatic activity when the reporter protein is fused with a large protein. Moreover, the methods fail to detect the type II membrane proteins having their N-terminus within the cell and C-terminus outside the cell.